The understanding of cellular functions relating to, for example, proliferation, migration, division, or differentiation requires an understanding of the mechanics of a cell's underlying cytoskeletal architecture. The architecture of the cytoskeleton is related to the cell's shape, which can be dictated by a cell's surrounding environment. Therefore, the cytoskeleton is in large part determined by a cell's responses to environmental stimuli.
Although various techniques have been developed to reproduce a cell's in vivo microenvironment, in general, it has not been possible to reproduce all of these aspects of a cell's in-vivo microenvironment within an in-vitro culture.
For example, Bhatia et al. (U.S. Patent Publication No. 2001/0023073) describe a hard, i.e., glass, substrate differentially functionalized through the aid of defined patterns developed using photoresist. Tan et al. (Tissue Eng., 2004, 10:865-72) describe differentially functionalized polydimethylsiloxane (PDMS) substrates. However, these substrates are hard substrates and uncharacteristic of living tissue.
Pelham and Wang (Proc. Natl. Acad. Sci. USA, 1997, 94: 13661-13665) describe a method of producing polyacrylamide cell substrates. However, these substrates are completely covered by a functionalizing linker molecule and, thus, do not replicate the cell's surrounding environment in vivo. Wang et al. (Cell Motil. Cytoskeleton, 2002, 52:97-106) describe a differentially functionalized polyacrylamide substrate developed using a first mask to deposit photoresist and subsequently producing a second mask of polymer (polydimethylsiloxane (PDMS)) to create a pattern on the substrate. Thus, this technique requires the production of multiple masks, and because the PDMS membranes must be sufficiently thick to be handled, and the channels sufficiently high to allow reasonable flow rates to functionalize the substrate, the spatial resolution, as well as the complexity, types and sizes of patterns are limited. Furthermore, since these polymer membranes are very thin, they are easily distorted, limiting the ability to precisely generate long, large patterns that are separated by spacing smaller than the length scale of the pattern.
Therefore, there exists a need in the art for methods for producing soft substrates that are tunable to mimic physiological tissues, that are differentially functionalized, and can be easily produced. In addition, such methods must allow for high fidelity pattern transfer and enhanced reproducibility.